Effects of oviductal and cumulus cells on in vitro
fertilization and embryo development of porcine
oocytes fertilized with epididymal spermatozoa
R. Romar
a,*, P. Coy
a, S. Ruiz
a, J. Gadea
a, D. Rath
b aDepartment of Physiology (Veterinary Physiology), Faculty of Veterinary Science,University of Murcia, 30071Murcia, Spain
bInstitute of Animal Science and Animal Behaviour, Mariensee, Germany
Received 2 May 2002; received in revised form 30 June 2002; accepted 2 July 2002
Abstract
This study was designed to evaluate the effects of adding porcine oviductal epithelial cell (POEC) monolayers before or during the fertilization of denuded or cumulus-enclosed oocytes, in terms of fertilization results and subsequent embryo development. The variables determined were: penetration rate, mean number of spermatozoa per oocyte, male pronucleus formation rate, monospermy rate, cleavage rate after 48 h of fertilization, blastocyst rate, and mean number of nuclei per blastocyst. We used cumulus-free and cumulus-enclosed oocytes preincubated or fertilized in the presence of POEC, once the purity in epithelial cells of these cultures had been assessed. All the experiments involved the use of frozen-thawed epididymal spermatozoa to avoid replicate variability. The POEC cultures prepared showed a high proportion of epithelial cells (over 95%). Preincubation of oocytes with POEC before fertilization showed no effects on the fertilization variables determined. In contrast, during IVF under our experimental conditions, these cells attached to the cumulus cells and their interaction had a signi®cant effect on some of the fertilization variables analyzed. The presence of POEC and cumulus cells during IVF increased oocyte penetrability. Moreover, in the absence of POEC, cumulus cells resulted in a reduced monospermy rate. On subsequent embryo culture, a lower cleavage and blastocyst formation rate were recorded when the oocytes had been preincubated with POEC before IVF.
#2002 Elsevier Science Inc. All rights reserved.
Keywords: Oviductal cells; Cumulus cells; Oocyte; Spermatozoa; IVF; Pig
*Corresponding author. Tel.:34-968-364-309; fax:34-9683-64147.
E-mail address:rromar@um.es (R. Romar).
0093-691X/02/$ ± see front matter#2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 1 1 3 8 - X
1. Introduction
For over a decade, oviductal cells have been used in culture systems in an effort to
approximate laboratory conditions to those encountered by the gametes in the oviduct[19].
In recent years both oviductal cells and products or conditioned media have been employed
by several research groups[5,11,13,19,28]. In pigs, the oviductal cells used are obtained
from different parts of the oviduct[6,7,29], at different stages of the estrous cycle[30,32]or
are introduced at different times during in vitro fertilization[28]. The effect of the presence
of these cells on penetrability by ejaculated spermatozoa (fresh or frozen-thawed) has been widely investigated and, in general, they increase penetrability and according to some
authors improve monospermy rates[6,19]. The use of oviductal cells before IVF has been
less widely explored in spite of being shown that the preincubation of pig oocytes with
oviductal cell monolayers[28]or with a porcine oviduct-speci®c glycoprotein[14]increases
the rate of monospermic fertilization. Unfortunately, this type of study has not been developed further and the possible in¯uence of these cells on subsequent embryo devel-opment remains to be established. Moreover, oviductal cells seem to show different behavior depending on the type of spermatozoa employed (fresh ejaculated versus cryopreserved
ejaculated) possibly modifying penetrability results[28]. Although there have been reports
of improved results related to the use of epididymal over ejaculated spermatozoa[25], there
is scarce reference to their use in the literature. This kind of semen have some advantages in front of the ejaculated one such as its simplicity to be frozen in small containers, a higher
motility and consistent in vitro fertilization rates with minimal variability[25].
Although it is widely accepted that cumulus cells are needed throughout the in vitro oocyte maturation process for appropriate cytoplasmic maturation facilitating subsequent
male pronucleus formation[9,16], their behavior during IVF is yet to be established. Under
in vivo conditions, porcine oocytes enter the oviduct and are fertilized while enveloped by their cumulus cells. In pig IVF systems, however, the use of cumulus-enclosed or denuded oocytes is not standardized and is usually determined by the kind of spermatozoa used:
refrigerated or frozen-thawed, respectively [2,10,12,35]. The effects of these cells on
penetrability by epididymal spermatozoa have not been widely investigated. Besides, these studies invariably involved stopping the process after fertilization so there is a lack of information on the subsequent effects on embryo development.
Insummary,althoughtheeffectsofbothoviductalandcumuluscellshavebeenexaminedin thepast,theirpossibleroleinIVFandearlyembryodevelopmentandtheirsuitabilityforusein IVF systems comprised of denuded or cumulus-enclosed oocytes and epididymal sperma-tozoa remain unclear. The present study was thus designed to evaluate the effects of porcine oviductal epithelial cell (POEC) monolayers added before and during IVF using denuded or cumulus-enclosed oocytes on fertilization results and subsequent embryo development.
2. Materials and methods 2.1. Culture media
Unless otherwise indicated, all the chemicals used in this study were purchased from Sigma±Aldrich QuõÂmica S.A. (Madrid, Spain). The medium used for oocyte maturation
was NCSU-37[22]supplemented with 0.57 mM cysteine, 1 mM dibutyryl cAMP, 5mg/ml
insulin, 50mM b-mercaptoethanol, 1 mM glutamine, 10 IU/ml eCG (Foligon, Intervet
International B.V., Boxmeer, Holland), 10 IU/ml hCG (Chorulon, Intervet International B.V., Boxmeer, Holland), 10 ng/ml EGF, and 10% (v/v) porcine follicular ¯uid.
The fertilization medium was modi®ed TALP[26] supplemented with 3 mg/ml fatty
acid-free BSA and 1.10 mmol/l Na-pyruvate. The medium for embryo culture was
NCSU-23[15]supplemented with 4 mg/ml fatty acid-free BSA and 1 mM glutamine.
Oviductal epithelial cells were cultured in TCM 199 supplemented with 13% (v/v) FCS,
150 IU/ml penicillin, and 100mg/ml streptomycin (Gibco BRL, Paisley, UK).
2.2. Oocyte collection and in vitro maturation
Ovaries from prepubertal gilts were transported to the laboratory in saline (0.9%, w/v
NaCl) containing 100 mg/ml kanamycin at 378C. Cumulus±oocyte complexes were
collected from non-atretic follicles (3±6 mm in diameter) by aspiration, and washed twice in modi®ed Dulbecco phosphate buffered saline (PBS) supplemented with 1 mg/ml polyvinyl alcohol. Oocytes with several layers of cumulus oophorus cells showing a homogeneous and granulated cytoplasm were selected and rinsed twice in maturation
medium, previously equilibrated for a minimum of 3 h under 5% CO2in air at 38.58C.
Groups of 50 oocytes were cultured in 500ml maturation medium for 22 h under 5% CO2
in air at 38.58C. After culture, the oocytes were washed three times and transferred to
hormone-free maturation medium for a further 22 h[8].
2.3. Culture of POEC
The procedure for culture of POEC was that described by Ouhibi et al.[20]with minor
modi®cations[28]. Oviducts from prepubertal gilts were recovered from the
slaughter-house and transported to the laboratory. They were then rinsed once in saline at 378C and
twice in PBS before being transferred to a Petri dish within a laminar ¯ow hood where fat pads and connective tissues were removed with sterile forceps and ®ne scissors. The oviducts were closed at one end with a clip, ®lled with a trypsin±EDTA solution for
endothelial cell culture, (500 BAAE units of porcine trypsin and 180mg EDTA) closed at
the other end and incubated for 45 min at 38.58C.
Following incubation, the wall of the oviduct was gently squeezed and its contents recovered in a Petri dish containing 10 ml preequilibrated culture medium. The epithelial cell clusters were dissociated by gentle, repeated pipetting followed by centrifugation at
800gfor 4 min. The resultant supernatant was discarded and the pellet resuspended in
fresh culture medium and seeded at a ®nal concentration of approximately 105cells/well.
The 4-well Nunc plates were maintained at 38.58C under 5% CO2and the medium was
changed after 48 h, and again every 2 days. Cells reached con¯uence after 5±7 days of initial seeding. Cell viability was directly evaluated by observing the cilial movement and progressive growth of epithelial cells.
Cell purity was checked by indirect immunocytochemistry with a monoclonal antibody
raised against cytokeratin[4,27]produced using bovine epidermal keratin as immunogen
times in PBS and then ®xed for 7 min in methanol/acetone (7:3, v/v). Next, 50ml of the anti-cytokeratin antibody 8.13 diluted 1:20 (v/v) in PBS was added. The cells were
incubated with the antibody for 1 h at 378C in a humidi®ed chamber and then washed three
times with PBS. Fifty microliters of a ¯uorescein isothiocyanate-labeled secondary antibody (Molecular probes, Leiden, Holland) diluted in PBS at 1:2000 (v/v) was then
added followed by incubation for 1 h in a humidi®ed atmosphere at 378C. The slides were
rinsed three times in PBS and ®nally covered with 100ml of Hoechst 33342 in methanol 1:100
(v/v). Labeled cells were visualized using an epi¯uorescence microscope (magni®cation
400) and those showing a ¯uorescent cytoplasm were considered to be epithelial cells.
2.4. In vitro fertilization and embryo culture
Frozen epididymal semen from one stud boar (Large WhiteLandrace) was used in all
the experiments. Prior to IVF, three 0.25 ml straws were thawed in warm water (20 s at
388C). After estimating sperm motility under a phase contrast microscope (magni®cation
200), the semen samples were diluted in Androhep1(MinituÈb, Tiefenbach, Germany)
and centrifuged at 800gfor 3 min. The sperm pellet was resuspended in modi®ed TALP
medium equilibrated overnight in an incubator at 38.58C under 5% CO2. In all the
experiments, the ®nal sperm concentration was adjusted to a ratio of 1500
spermato-zoa:oocyte, and 10ml of diluted spermatozoa were added to the plates containing the
oocytes equilibrated for 30 min before introducing the sperm.
After an IVF period of 18 h, presumptive zygotes were cultured in groups of 40
maximum for an additional 7 days in 2 ml NCSU-23 medium [23]. The medium was
replaced once after 2 days of culture.
2.5. Assessment of fertilization results and developmental capacity of embryos
After the IVF period, a representative sample of each group was ®xed in acetic acid± ethanol (1:3, v/v), stained with 1% (w/v) lacmoid after a minimum of 24 h, and examined
under a phase contrast microscope (magni®cation 400) for evidence of sperm penetration
and pronucleus formation.
Forty-eight hours after insemination, the cleavage of oocytes was evaluated under a
stereomicroscope equipped with a warm plate at 38.58C and a special chamber connected to
a 5% CO2permanent supply tube. Two 4-cell stage embryos were recorded as cleaved.
Blastocysts were assessed on day 7 by observation of a clear blastocele under the stereo-microscope. Nuclei were counted by ®xing and staining with Hoechst 33342 1% in PBS.
2.6. Experimental design
Before the in vitro use of POEC, we conducted a pilot experiment to test the purity of the epithelial cell cultures. Oviductal cells were cultured as previously described and subjected
to four replicate immunocytochemical tests [4].
In the Experiment 1, we investigated the effects of preincubating denuded and cumulus-enclosed oocytes with POEC before IVF on fertilization results and embryo development. After the IVM period, half of a group of oocytes was preincubated for 3 h with oviductal
epithelial cell monolayers preequilibrated for 1 h in modi®ed TALP medium (POEC preincubation group) and the other half was maintained in maturation medium (non-preincubated oocyte group). The oocytes used were cumulus-enclosed or were denuded by pippeting. After the 3 h, the non-preincubated oocytes were denuded or not and then all the
oocytes were fertilized in the absence of oviductal cells. Oocytes were transferred to 90ml
of TALP medium covered with paraf®n oil and after 30 min at 38.58C and 5% CO2, 10ml
of sperm solution was introduced. After IVF, a subsample of 15 oocytes from each group was ®xed and stained to assess the fertilization results, and the remaining zygotes were transferred to embryo culture. Cleaved embryos, blastocyst formation, and number of nuclei per blastocyst were recorded 2 and 7 days after fertilization, respectively. This experiment was performed as six replicates and the raw data were pooled.
Experiment 2 was designed to evaluate the effects of adding POEC during the IVF of cumulus-free and cumulus-enclosed oocytes on fertilization and embryo development variables. After IVM, half of the oocytes were denuded by pipetting and the other left intact with their cumulus cells. These oocytes were then washed twice in modi®ed TALP medium, transferred to a 4-well plate and fertilized in the presence (POEC IVF group) or absence (POEC-free IVF group) of a monolayer of oviductal cells equilibrated for 1 h in
190ml of TALP. After the IVF period, a subsample of 15 oocytes from each group was ®xed
and stained to assess the fertilization results and the remaining zygotes were transferred to an embryo culture medium. Cleaved embryos and the rate of blastocyst formation and number of nuclei per blastocyst were recorded 2 and 7 days after fertilization, respectively. This experiment was performed as ®ve replicates and raw data were pooled.
2.7. Statistical analysis
Data are presented as the meanS:E:M:, and all rates were modeled according to the
binomial model of parameters. The variables, oocyte penetration rate, number of sperm cells per penetrated oocyte, male pronucleus formation and monospermy rates, as well as cleavage and blastocyst formation rates, and the number of cells/blastocyst were analyzed by two-way ANOVA, considering the presence of POEC and cumulus cells as main effects. When a signi®cant effect was revealed by ANOVA, values were compared using the Tukey
test. AP value <0.05 was taken to denote statistical signi®cance.
3. Results
The immunocytochemistry test con®rmed that 99:301:85% of the oviductal cells
grown in vitro were epithelial, showing a positive staining with the anti-cytokeratin 8.13
antibody (Fig. 1).
3.1. Experiment 1
Preincubating the oocytes with oviductal epithelial cells for 3 h before IVF showed no
effect on any of the fertilization variables determined (Table 1). Cumulus-enclosed oocytes
exclusively on the group of POEC preincubated oocytes, penetration rates were similar for the denuded oocytes and those surrounded by their cumulus cells (93.51 versus 92.22). The presence of cumulus cells led to a signi®cantly increased mean number of sperm per oocyte, both in the POEC preincubated oocyte and in the non-preincubated control groups. However, no effect of cumulus cells was observed on male pronucleus formation after IVF, which was over 90% in all the groups.
Fig. 1. (a) Isolated oviductal cells attached to the bottom of the Petri dish after 72 h of culture. (b) Oviductal cells after 5 days of culture showing a non-covered area. (c) Con¯uent monolayer of POECs after 7 days of culture. (d) POECs. Immunostaining with anti-cytokeratine 8.13 showing the positive staining of the cytoplasm.
Table 1
Effect of preincubating oocytes with POEC on the fertilization of cumulus-enclosed and denuded oocytes POEC preincubation Cumulus N Penetration (%) S/O % MPN1 Monospermy (%)1
No No 83 83.134.14a 2.100.14a 95.652.47 43.486.01a Yes 81 96.302.11b 4.500.24b 98.721.28 6.412.79b Yes No 77 93.512.83ab 2.510.17a 91.673.28 25.005.14c Yes 77 92.223.07ab 4.080.26b 97.181.98 11.273.78bc Source of variability POEC preincubation 0.319 0.994 0.238 0.132 Cumulus 0.060 <0.001 0.067 <0.001 POEC preincubation cumulus 0.022 0.056 0.600 0.010
N, number of oocytes;S/O, mean number of spermatozoa per penetrated oocyte; and % MPN, male pronucleus formation rate. Values with different superscripts (a, b, c) within columns are signi®cantly different (P<0:05).
In contrast, the presence of cumulus cells had a detrimental effect on monospermy rates
(P<0:001), best results being achieved with denuded oocytes. This effect lost its
signi®cance, however, when the oocytes were preincubated with oviductal cells before IVF (25 versus 11.27).
The embryo development results (Table 2) indicate that embryos formed from
non-preincubated oocytes showed a signi®cantly higher cleavage rate than those derived from
POEC preincubated oocytes (P0:019). The presence of cumulus cells showed no effect
on this variable. Preincubation with POEC had a negative effect on the blastocyst formation
rate (P<0:001) and this trend was also related to the presence of cumulus cells
(P0:056). Thus, highest blastocyst formation rates were achieved after the IVF of
non-POEC preincubated, denuded oocytes.
Finally, neither POEC preincubation nor cumulus cell presence had an effect on the ®nal quality of the blastocyst, assessed by the mean number of cells per blastocyst, which was similar in each group.
3.2. Experiment 2
The presence of a monolayer of oviductal cells during the fertilization of oocytes with
epididymal spermatozoa signi®cantly increased penetrability (Table 3), both in terms of the
penetration rate (P0:010) and the mean number of sperm per oocyte (P<0:001).
Cumulus-enclosed oocytes showed a tendency towards easier penetrability than
denuded oocytes (P0:054). A signi®cant effect on the mean number of spermatozoa
was observed (P0:027), highest values being recorded for oocytes fertilized in the
presence of both types of cells (3.29 spermatozoa/oocyte). The latter was signi®cantly different from that noted for oocytes fertilized in the absence of POEC.
As in Experiment 1, neither the presence of oviductal or cumulus cells during IVF affected male pronucleus formation, which again was around 90% in each group. Similarly, no effects of either cell type were observed on the ®nal rates of monospermy, which ranged
Table 2
Effect of preincubating oocytes with POEC before IVF on embryo development of cumulus-enclosed and denuded oocytes
POEC preincubation Cumulus N Cleavage (%) Blastocysts (%)1 Ncells/blastocyst
No No 360 80.562.09 30.692.71a 20.471.17 Yes 383 80.422.03 21.432.34b 18.171.26 Yes No 317 74.132.46 17.452.48b 20.041.49 Yes 322 76.402.37 17.072.40b 19.472.01 Source of variability POEC preincubation 0.019 <0.001 0.784 Cumulus 0.633 0.056 0.366 POEC preincubation cumulus 0.590 0.078 0.583
Nis the number of zygotes. Values with different superscripts (a, b) within columns are signi®cantly different (P<0:05).
from 21.62 to 35.71%. This last value corresponds to oocytes fertilized in the absence of both oviductal and cumulus cells.
The embryo culture data (Table 4) indicate that the presence or absence of oviductal cells
during IVF failed to affect any of the embryo development variables examined, although
there was a tendency towards higher cleavage rates (P0:07) when these cells were
present. In contrast, cumulus cells showed a positive effect on cleavage rate (P0:003)
but not on the blastocyst formation rate.
4. Discussion
The immunocytochemical detection of keratins is the most commonly used method of
characterizing epithelial cell cultures[4,27]. In our study, this technique served to con®rm
Table 3
Effect of POEC monolayers on the fertilization of cumulus-enclosed and denuded oocytes
POEC IVF Cumulus N Penetration (%) S/O % MPN1 Monospermy (%)1
No No 73 76.714.98a 2.120.19a 89.294.17 35.716.46 Yes 70 88.573.83ab 2.330.15a 93.553.15 30.655.90 Yes No 78 91.033.26b 2.590.20ab 91.553.32 30.995.53 Yes 79 93.672.76b 3.290.24b 93.242.94 21.624.82 Source of variability POEC 0.010 <0.001 0.772 0.225 Cumulus 0.054 0.027 0.379 0.202 POECcumulus 0.220 0.237 0.704 0.704
N, number of oocytes; S/O, mean number of spermatozoa per penetrated oocyte; and % MPN, male pronucleus formation rate. Values with different superscripts (a, b) within columns are signi®cantly different (P<0:05).
1With respect to the number of penetrated oocytes.
Table 4
Effect of POEC monolayers during the IVF of cumulus-enclosed and denuded oocytes on embryo development POEC IVF Cumulus N % Cleavage Blastocysts (%)1 Ncells/blastocyst
No No 320 59.372.75a 21.582.99 22.651.98 Yes 305 71.482.59b 17.892.60 19.001.21 Yes No 349 68.482.49ab 14.642.29 20.001.74 Yes 288 71.872.65b 16.912.61 16.941.15 Source of variability POEC 0.070 0.130 0.160 Cumulus 0.003 0.785 0.046 POECcumulus 0.098 0.255 0.858
Nis the number of zygotes. Values with different superscripts (a, b) within columns are signi®cantly different (P<0:05).
the purity of our oviductal cell cultures in epithelial cells (over 95%) and the lack of contamination with other cell types. These ®ndings are comparable to those reported by
others for bovine cell cultures[27,31,33].
The use of oviductal cells for oocyte preincubation before IVF and during IVF using
ejaculated spermatozoa[11,19]has been associated with improved rates of monospermy.
In the present study, the preincubation of oocytes with POEC for 3 h before IVF showed
no effect on the fertilization variables recorded (Table 1). Possible explanations for this
could be an insuf®ciently long preincubation time to considerably affect the oocyte, the incapacity of the IVF system to discriminate possible effects (given the high sperma-tozoa:oocyte ratio) or that the POEC culture employed was not ``functional'' or ``active.'' In our opinion, any of these three hypotheses might be valid. Indeed, bene®cial effects on monospermy have been described after the preincubation of porcine oocytes with POEC
[28]or puri®ed porcine glycoproteins[14]. In both these studies, the contact time ranged
from 2 to 4 h before IVF and ejaculated spermatozoa were used. Some authors propose that the effect of the oviductal cell disappears or is masked when high sperm
concentra-tions are used[5,28]. Finally, with regard to the functionality of the culture, it has been
shown that oviductal cells treated with estradiol give rise to improved monospermy rates
over non-treated or progesterone-treated cells[6]. In our study, we were able to con®rm
that the oviductal cell cultures used were mainly epithelial but we have no information on their ``functionality.'' The fact that preincubation with POEC also failed to affect the rate of male pronucleus formation is in agreement with the observations of other researchers
working with preincubated oocytes[14,28]suggesting that the cytoplasmic maturation
of oocytes is suf®cient to achieve a high rate of pronucleus formation after IVM in NCSU-37.
Cumulus cells have been described to show a positive effect on penetrability by
ejaculated frozen-thawed spermatozoa [35,36] and were associated here with the
penetration of the oocyte by a greater number of epididymal spermatozoa. For different
species[3,17]several authors have referred to a bene®cial effect of the cumulus on the
acrosome reaction and this could account for the higher penetrability of the cumulus-enclosed oocytes we observed. Nevertheless, this enhanced penetrability led to a diminished monospermy that was signi®cant in the case of the oocytes not preincubated with POEC. Frozen-thawed spermatozoa show reduced motility after thawing but those able to cross the mass of cumulus cells before completely loosing their motility can maintain their activity and undergo capacitation and subsequently penetrate the oocyte
[34]. According to our results, this effect of cumulus cells also appears to apply to
epididymal spermatozoa and is in line with previous ®ndings[12]. The dependence on
cumulus cells of frozen boar epididymal spermatozoa to complete capacitation was
suggested by Nagai et al.[18]. However, the results of both our experiments indicate that
with the system employed, high penetration rates of denuded oocytes can also be achieved using epididymal spermatozoa.
It should be noted that the oocytes preincubated with POEC before IVF showed similar penetration rates, irrespective of the presence or absence of cumulus cells (93.51 versus
92.22,Table 1). This could be attributable to some kind of interaction between these two
cell types leading to alterations at a level (e.g. the zona pellucida) not re¯ected by conventional fertilization variables.
As some authors have already indicated, cleavage rate does not objectively assess the
embryo culture system[1]. Moreover, a high proportion of in vitro produced pig embryos
have some kind of morphological abnormality and a low number of cells[37]. Here, as a
logical consequence of the rate of monospermy, according to the blastocyst formation rate, non-POEC preincubated, denuded oocytes showed the highest output, although the mean number of cells/blastocyst was similar between groups and within the range quoted by
other authors[24,26].
In the second experiment, oocytes subjected to IVF in the presence of POEC showed
increased penetrability (Table 3). These same observations were previously associated with
the use of ejaculated spermatozoa, both refrigerated[11,19]and frozen-thawed[21,28]but,
to our knowledge, our results are the ®rst to refer to the use of epididymal spermatozoa. This observation could be interpreted as the effect of the oviductal cells on the spermatozoa favoring sperm capacitation, since in this experiment, unlike the previous one, the monolayers were present for the entire 18 h of contact between gametes. These data
would be in agreement with those reported by Fazeli et al. [7], who observed sperm
capacitation induced by oviductal cells in boar ejaculated spermatozoa. However, the presence of POEC during IVF did not signi®cantly affect male pronucleus formation (Table 3) in agreement with other papers[11,19]. As in Experiment 1, although perhaps not as evident, cumulus-enclosed oocytes presented a higher number of sperm per oocyte than their denuded counterparts. These data would also appear to point to a bene®cial effect of cumulus and oviductal cells on sperm capacitation and the acrosome reaction or, at least, on oocyte penetrability.
The effects observed on the variables of fertilization outcome could not be extrapolated to subsequent embryo development since no effect was shown by the presence of POEC during IVF on the cleavage and blastocyst rates or mean number of cells per embryo (Table 4). Although in this experiment the presence of cumulus cells during IVF did have a signi®cant effect on the cleavage rate, in our opinion we should be cautious in our interpretation because of the subjectivity involved in evaluating this variable as mentioned previously. The proof of this is that the rate of blastocyst formation showed no effect of the cumulus and is, no doubt, a consequence of the cleavage rate.
In conclusion, our data show that the preincubation of oocytes with POECs 3 h before IVF does not affect the typically estimated fertilization variables and that the copresence of these and cumulus cells during IVF with epididymal spermatozoa increases oocyte penetrability. Finally, under the experimental conditions employed neither POEC preincubation nor presence of POEC during IVF improved the embryo development results.
Acknowledgements
The authors would like to thank Dr. Wilfrid Kues (Institute of Animal Science and Animal Husbandry, Mariensee, Germany) for his experienced assistance in the immuno-cytochemistry test, and Mrs. Antje Frenzel and Mrs. Birgit Sieg for their valuable help in the laboratory. This research was supported by projects 1FD97-501, HA99-115 (DAAD 314), and AGL 2000-0485-C02-01.
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